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Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.

Koprich JB, Johnston TH, Reyes MG, Sun X, Brotchie JM - Mol Neurodegener (2010)

Bottom Line: The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01).At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Toronto Western Research Institute, Toronto Western Hospital, University Health Network, 399 Bathurst Street, Toronto, ON, M5T 2S8, Canada. jkoprich@uhnres.utoronto.ca.

ABSTRACT

Background: The pathological hallmarks of Parkinson's disease (PD) include the presence of alpha-synuclein (α-syn) rich Lewy bodies and neurites and the loss of dopaminergic (DA) neurons of the substantia nigra (SN). Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV), or green fluorescent protein (GFP), the latter to control for the possibility that high levels of protein in themselves might contribute to damage.

Results: We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01). At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.

Conclusions: In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model. The dynamics of the evolution of the pathology however, provide advantages over current models with respect to providing an initial screen to assess efficacy of novel treatments that might prevent/reverse α-syn aggregation.

No MeSH data available.


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Tyrosine hydroxylase and NeuN cell counting in the substantia nigra. Three weeks following delivery of AAV1/2 to the substantia nigra there is significant reductions in the number of TH-immunoreactive neurons and NeuN-immunoreactive neurons for both alpha-synuclein and GFP treated animals compared to empty vector controls. AAV1/2 A53T alpha-synuclein produced significantly greater TH and NeuN cell loss compared to the GFP group. *P < 0.05 cf. empty vector; ** P < 0.01 cf. empty vector;*** P < 0.001 cf. empty vector; ## P < 0.01 cf. GFP; scale bar is 1000 μm.
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Figure 4: Tyrosine hydroxylase and NeuN cell counting in the substantia nigra. Three weeks following delivery of AAV1/2 to the substantia nigra there is significant reductions in the number of TH-immunoreactive neurons and NeuN-immunoreactive neurons for both alpha-synuclein and GFP treated animals compared to empty vector controls. AAV1/2 A53T alpha-synuclein produced significantly greater TH and NeuN cell loss compared to the GFP group. *P < 0.05 cf. empty vector; ** P < 0.01 cf. empty vector;*** P < 0.001 cf. empty vector; ## P < 0.01 cf. GFP; scale bar is 1000 μm.

Mentions: Three weeks following surgical delivery of AAV1/2 vectors to the rat SN there were significant differences in the amount of TH-immunoreactive neurons present quantified using unbiased stereology [F(2,12) = 26.15, P < 0.0001] (Figure 4A-D). To serve as a control, identical AAV1/2-empty vectors were delivered in the same manner and concentration. Post-hoc analysis revealed that rats that received AAV1/2-A53T α-syn had significantly less TH-immunoreactive neurons compared to those injected with either AAV1/2-empty vector (52% reduction, P < 0.001) and GFP (37% reduction, P < 0.01). Furthermore, there were significantly less TH-immunoreactive neurons in rats receiving AAV1/2-GFP compared to those that received AAV1/2-empty vector controls (24% reduction, P < 0.05). To confirm that the AAV1/2-EV was not toxic to DA neurons we counted TH-immunoreactive neurons in the SN opposite to the injected side and showed that there was no significant difference between hemispheres (t[8] = 1.09, P > 0.05).


Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.

Koprich JB, Johnston TH, Reyes MG, Sun X, Brotchie JM - Mol Neurodegener (2010)

Tyrosine hydroxylase and NeuN cell counting in the substantia nigra. Three weeks following delivery of AAV1/2 to the substantia nigra there is significant reductions in the number of TH-immunoreactive neurons and NeuN-immunoreactive neurons for both alpha-synuclein and GFP treated animals compared to empty vector controls. AAV1/2 A53T alpha-synuclein produced significantly greater TH and NeuN cell loss compared to the GFP group. *P < 0.05 cf. empty vector; ** P < 0.01 cf. empty vector;*** P < 0.001 cf. empty vector; ## P < 0.01 cf. GFP; scale bar is 1000 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984491&req=5

Figure 4: Tyrosine hydroxylase and NeuN cell counting in the substantia nigra. Three weeks following delivery of AAV1/2 to the substantia nigra there is significant reductions in the number of TH-immunoreactive neurons and NeuN-immunoreactive neurons for both alpha-synuclein and GFP treated animals compared to empty vector controls. AAV1/2 A53T alpha-synuclein produced significantly greater TH and NeuN cell loss compared to the GFP group. *P < 0.05 cf. empty vector; ** P < 0.01 cf. empty vector;*** P < 0.001 cf. empty vector; ## P < 0.01 cf. GFP; scale bar is 1000 μm.
Mentions: Three weeks following surgical delivery of AAV1/2 vectors to the rat SN there were significant differences in the amount of TH-immunoreactive neurons present quantified using unbiased stereology [F(2,12) = 26.15, P < 0.0001] (Figure 4A-D). To serve as a control, identical AAV1/2-empty vectors were delivered in the same manner and concentration. Post-hoc analysis revealed that rats that received AAV1/2-A53T α-syn had significantly less TH-immunoreactive neurons compared to those injected with either AAV1/2-empty vector (52% reduction, P < 0.001) and GFP (37% reduction, P < 0.01). Furthermore, there were significantly less TH-immunoreactive neurons in rats receiving AAV1/2-GFP compared to those that received AAV1/2-empty vector controls (24% reduction, P < 0.05). To confirm that the AAV1/2-EV was not toxic to DA neurons we counted TH-immunoreactive neurons in the SN opposite to the injected side and showed that there was no significant difference between hemispheres (t[8] = 1.09, P > 0.05).

Bottom Line: The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01).At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Toronto Western Research Institute, Toronto Western Hospital, University Health Network, 399 Bathurst Street, Toronto, ON, M5T 2S8, Canada. jkoprich@uhnres.utoronto.ca.

ABSTRACT

Background: The pathological hallmarks of Parkinson's disease (PD) include the presence of alpha-synuclein (α-syn) rich Lewy bodies and neurites and the loss of dopaminergic (DA) neurons of the substantia nigra (SN). Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV), or green fluorescent protein (GFP), the latter to control for the possibility that high levels of protein in themselves might contribute to damage.

Results: We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01). At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.

Conclusions: In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model. The dynamics of the evolution of the pathology however, provide advantages over current models with respect to providing an initial screen to assess efficacy of novel treatments that might prevent/reverse α-syn aggregation.

No MeSH data available.


Related in: MedlinePlus